This invention relates to improved methods for manufacturing extremely thin, very delicate metallic structures possessing grid-like patterns of minute, closely spaced, precisely dimensioned apertures. Such apertured metal structures, hereinafter referred to as "microsieves", are especially useful in sorting and sieving objects of only a few microns in size. One such microsieve, designated a "cell carrier", is described in Spanish patent No. 522,207, granted June 1, 1984, and in commonly assigned, copending U.S. patent application Ser. No. 550,233, filed Nov. 8, 1983, the disclosure of which is incorporated by reference herein, for classifying biological cells by size. The cell carrier is prepared employing a modified photo-fabrication technique of the type used in the manufacture of transmission electron microscope grids. The cell carrier is on the order of only a few microns in thickness and possesses a numerically dense pattern of minute apertures. Even with the exercise of great care, the very delicate nature of the cell carrier makes it difficult to manipulate, for example, to insert it in a holder of the type shown in aforesaid U.S. patent application Ser. No. 550,233, without causing it appreciable damage, frequently in the form of a structural deflection or deformation which renders it useless for its intended use.
In order to better understand and appreciate the improvements and advantages made possible by the present invention, the foregoing known type of microsieve, or cell carrier as it is called, and a method for its manufacture will be described in connection with the accompanying figures of drawing, all of which are greatly enlarged in size and with certain features exaggerated for the sake of clarity, in which FIG. 1(a) is a plan view of the cell carrier, FIGS. 1(b) and 1(c) are perspective and side elevational views, respectively, of a typical secticn of the cell carrier and FIGS. 2(a) through 2(e) are side elevational views of successive steps in the manufacture of a section of the cell carrier.
The cell carrier 10 shown in FIG. 1(a) is a very thin metallic disk, for example, about 8 to 10 microns in thickness, with a square-shaped, grid-like pattern of apertures 11 with centers about 15 microns apart defined within its geometric center. The cell carrier can be fabricated from a variety of metals including copper, nickel, silver, gold, etc., or a metal alloy. The apertures actually number 100 on a side for a total of 10,000 apertures and are thus able to receive, and retain, up to 10,000 cells of the desired size with each cell occupying a single aperture. Keyway 12 is provided to approximately orient the cell carrier within its holder.
As shown in FIGS. 1(b) and 1(c), a representative section of grid 11 of cell carrier 10 possesses numerous apertures or holes 20 arranged in a matrix-like pattern of rows and columns along axes X and Y respectively. This arrangement makes it possible to label and locate any one aperture in terms of its position along coordinates X and Y. The shape of apertures 20 enables biological cells 21 of preslected dimensions to be effectively held to the carrier by applying means, such as a pressure differential between the upper and the bottom side of the carrier, or electromagnetic forces. To first separate a particular group of cells from cells of other groups, carrier 10 is chosen to have apertures of sizes so that when the matter, for example, blood, containing the various cell groups is placed on carrier 10, most, if not all, of the apertures become occupied by cells of the group of interest with each aperture containing one such cell. Thus, the apertures can be sized to receive, say, lymphocytes of which there are two principal sizes, namely, those of 7 microns and those of 10-15 microns, with the former being the cells of most interest and the latter being washed away from the upper surface 10t of the grid under a continuous flow of fluid. To capture and retain the smalle size lymphocytes, apertures 20 will have an upper cross-sectional diameter of about 6 microns and a lower cross-sectional diameter of about 2 microns or so. In this way, a lymphocyte from the desired population of cells can easily enter an aperture but once it has occupied the aperture, it cannot pass out through the bottom side 10b of the carrier. The cut-out areas 30(d) about the bottom of each aperture have no functional significance and result from the procedures whereby the cell carrier is manufactured as discussed below in connection with FIGS. 2(a) through 2(e).
In the initial steps of the known method of manufacturing cell carrier 10 which are illustrated in FIGS. 2(a) through 2(e), a layer of photoresist 30, e.g., a photoemulsion, having a thickness, or height, generally on the order of about 1 micron or so, is applied to a metallic base plate, or mandrel, 31, e.g., of copper, upon which the carrier is to be formed. In FIG. 2(b), photoemulsion layer 30 has been selectively exposed to a source of actinic radiation employing a conventional mask procedure to produce a patterned surface of discrete areas of unexposed photoemulsion 30(a) surrounded by a continuous area 30(b) of exposed photoemulsion. Following conventional treatment of photoemulsion layer 30 with developer, fixer and finally, with clearing agent to wash away exposed area 30(b), there remains discrete areas of fixed photoemulsion 30(a) supported upon mandrel 31 as shown in FIG. 2(c). These fixed areas of photoemulsion correspond to the sites later defining the bottoms of apertures 20 in the finished carrier 10 and most frequently will be circular in cross-section. As shown in FIG. 2(d), a continuous layer of metal 30(c), e.g., copper, gold, nickel, silver, etc., or metal alloy, which is to provide the body of cell carrier 10, is electrodeposited upon mandrel 31. Since fixed areas 30(a) of the photoemulsion 10 are very thin, in order to build up the thickness of the carrier, or aperture height, some of metal 30(c) will inevitably overflow onto the peripheral edges of fixed areas 30(a) to form an aperture having a cone-shaped bore. Clearly, as one increases the thickness of the electrodeposited metal, the steeper will be the slope of the ultimate aperture bore. To prevent the aperture from becoming occluded by the overflow of electrodeposited metal, it is necessary to place the areas of fixed photoemulsion further apart as the thickness (i.e., the height) of electrodeposited metal layer 30(c) is increased. This has the necessary consequence of reducing the number of apertures which can be formed in the metal structure as its thickness is increased. In the final manufacturing steps shown in FIG. 2(e), mandrel 31 is removed and the fixed areas 30(a) of the photoemulsion are dissolved, or etched, away to provide carrier 10 containing the desired pattern, or grid, of apertures 20. A circumferential cut-away area 30(d) which possesses no role in the operation of the cell carrier is defined in the bottom of each aperture once fixed photoemulsion areas 30(a) are removed.
The aforedescribed method for making a microsieve is subject to a number of disadvantages, foremost among them being the practical difficulty of providing a sufficient thickness, or aperture height, without simultaneously unduly reducing the numerical density of the apertures. In addition, because of the thinness of the microsieve (typically weighing about 400 micrograms or so) which is obtainable by this manufacturing method, the structure is mechanically very fragile and as a result, is difficult to manipulate without causing it to be distorted or damaged. Still another disadvantage lies in the fact that the sloping sides of apertures 20 make it easy for them to be occupied by more than one cell. Ideally, an essentially vertical slope is desired to prevent or minimize this possibility; however, such a slope cannot be obtained with the foregoing method.
Other prior art which may relate to one or more features of the present invention can be found in U.S. Pat. Nos. 2,968,555; 3,139,392; 3,190,778; 3,329,541; 3,403,024; 4,058,432; 4,388,351; and 4,415,405.